Genetic engineering utilizes systems of eukaryotic viral vectors to introduce exogenous DNA into animal or plant cells by transduction. The principal eukaryotic viruses which are likely to serve as DNA transduction vectors in mammalian cells (SV40 and polyoma, for example) and in plant cells (cauliflower mosaic virus, for example) can receive only exogenous ("foreign") DNA of restricted length due to the morphology of the structure of their nucleocapsid. Because the introduction of exogenous DNA could increase the viral genome beyond a size which could be packaged into the viral capsid, it has proved absolutely necessary to have available vectors capable of accepting more exogenous "foreign" DNA, especially since developments in genetic engineering are tending towards attempts to insert more than one foreign gene into a host-cell with the purpose, for example, of obtaining coordinated expression and, if possible, coordinated activity of the products of the foreign genes.
The ideal viral vector must also permit the introduction of lengthy foreign DNA segments into cells at high frequency, with efficient expression of one or more foreign genes.
Virus which seem to fulfill these conditions are the following baculoviruses: nuclear polyhedrosis virus and Autographa californica (AcNPV). L. K. Miller [Chapter 14 in "A Virus Vector for Genetic Engineering in Invertebrates" of the manual "GENETIC ENGINEERING IN PLANT SCIENCES" (1981) N. J. Panopoulos, Ed., Praeger Publ. New-York, pages 203-223] identifies the latter as possessing features potentially useful for use as a vector for the propagation and expression of foreign genes in an eukaryotic environment. See also an earlier article by K. M. Potter and L. K. Miller [ANIMAL VIRUS GENETICS, 6, GENETIC MUTATIONS OF A BACULOVIRUS, ACADEMIC PRESS (1980), pages 71-80].
A number of other publications are also directed to the use of baculovirus systems: European patent application Nos. 0127 839, 0228 036 and 0260 090. These three publications seem to have the common feature of requiring first the creation of a transfer vector, which is a construct capable of accepting a foreign gene, to in effect transfer the foreign gene as a separate step into the virus, a lengthy and delicate process.
An article by Smith, et al., ["PHYSICAL ANALYSIS OF AUTOGRAPHA CALIFORNICA NUCLEAR POLYHEDROSIS VIRUS TRANSCRIPTS FOR POLYHEDRIN AND 10000-MOLECULAR WEIGHT PROTEIN", Journal of Virology, (January 1983), pages 215-225] describes the approximate location of the DNA sequences which encode the mRNAs for the polyhedrin and p10 proteins of the AcNPV.
This invention has as its object a modified baculovirus bearing a unique restriction site situated downstream of the ATG codon of a gene encoding for at least one baculovirus-associated protein or downstream of a strong late promoter of the gene for a baculovirus-associated protein, suitable for use in inserting operatively heterologous DNA encoding a desired polypeptide.
This invention also has as it object the process of direct insertion of at least one heterologous DNA sequence into the above-described baculovirus expression vector by in vitro manipulation thus obviating the need for intermediate transfer vectors to achieve said insertion of heterologous DNA.
This invention further has as its object a process for the expression of foreign genes inserted into said baculovirus expression vectors.